Tissue-type plasminogen activator (tPA) is one of the major components in the matrix proteolytic network whose role in the pathogenesis of renal interstitial fibrosis remains largely unknown. Here, we demonstrate that ablation of tPA attenuated renal interstitial fibrotic lesions in obstructive nephropathy. Mice lacking tPA developed less morphological injury and displayed a reduced deposition of interstitial collagen III and fibronectin as well as total tissue collagen in the kidneys after sustained ureteral obstruction, when compared with their wild-type counterparts. Deficiency of tPA selectively blocked tubular epithelial-to-myofibroblast transition (EMT), but did not affect myofibroblastic activation from interstitial fibroblasts. A marked decrease in matrix metalloproteinase-9 (MMP-9) induction was found in the obstructed kidneys of tPA(-/-) mice, which led to a dramatic preservation of the structural and functional integrity of tubular basement membrane (TBM). In vitro, tPA induced MMP-9 gene expression and protein secretion in renal interstitial fibroblasts. Thus, increased tPA is detrimental in renal interstitial fibrogenesis through a cascade of events that lead to MMP-9 induction, TBM destruction, and promotion of EMT. Our findings establish a crucial and definite importance of EMT in the pathogenesis of renal interstitial fibrosis at the whole-animal level.